Letter to the editor: Low sensitivity of RT‐Alu‐PCR in detection of HBV integrated chimeric transcripts
Authors: Abudurexiti Abulaiti, Zhiqiang Gu, Qianqian Jiang, Hongxin Huang, Fengmin Lu
Journal: Hepatology (2022)

To the editor, We read with great interest the study by Erken et al. in which they developed Alu‐PCR and RT‐Alu‐PCR for quantitative detection of integrated HBV DNA and their chimeric transcripts, respectively.1 Because their detection of chimeric transcripts relied on the presence of the Alu sequence, here we demonstrate that a huge amount of chimeric transcripts would not be detected by RT‐Alu‐PCR. Firstly, up to 468 unique HBV integration sites were identified from two RNA‐sequencing (RNA‐seq) data sets (GSE94660 and SRA447498) of liver samples derived from 47 HBV‐related patients with HCC, through the Viral Integration Site Database. Among them, 344 unique long chimeric transcripts were reconstructed using transcriptome assembly tools. Unexpectedly, only 53 (15%) chimeric transcripts contained Alu elements (Figure 1A–C), indicating that nearly 85% of the chimeric transcripts would be missed by Erken et al. in their study.FIGURE 1: Low proportion of chimeric transcripts containing Alu elements. (A) The unique HBV integration sites in two RNA‐seq data sets of liver samples from 47 HBV‐related patients with HCC were identified, and the integrated long chimeric transcripts were reconstructed by trinity v2.14.0. (B) Representative screenshots of the integrative genomics viewer (IGV) showing the reconstructed chimeric transcripts in one patient with HBV‐related HCC with an Alu element. (C) Representative screenshots of the IGV showing the reconstructed chimeric transcripts in the same patient with HBV‐related HCC (B) without an Alu element. (D) Schematic diagram of HBV transcripts sharing the overlapping 3′ end.It is well known that all HBV covalently closed circular DNA transcripts share an overlapping 3′ end, making sequences of X transcripts completely contained within those of the S transcripts (Figure 1D). Theoretically, transcripts detected by RT‐Alu‐PCR targeting the X region would have a relatively higher level than those detected by targeting the S region. The inconsistent result observed by Erken et al. (figure 2 of their study) could be explained by the instability and inward shortening of the integrated HBV fragments along with disease progression,2,3 making it impossible to detect the 3′ truncated chimeric S and X transcripts. Indeed, levels of integrated HBV DNA containing open reading frame (ORF) X (but not S) in the chronic HBV‐infected individuals (ChI) group were lower than those in the HBeAg‐negative chronic hepatitis B (CH) group (figure 4A,D of their study).1 Meanwhile, though the integrated HBV DNA contained similar amounts of ORF S (figure 4A of their study), the phenomenon of fewer S transcripts in the ChI group than the HBeAg‐negative CH group could be attributed to epigenetic modification of integrated fragments, as reported.4 Alternatively, the loss of HBV enhancer II during the immune‐mediated shortening of integrated fragments could also be involved. In line with this, the author reported that the loss of integrated HBV DNA containing ORF S showed a relatively higher odds ratio than ORF X in predicting serum HBsAg loss during the follow‐up after standard pegylated interferon alpha treatment.